1. Field of the Invention
This invention relates to a cell analyzing apparatus of the type which employs flow cytometry.
2. Description of the Related Art
In flow cytometry, a sample containing cells (or particles that are the equivalent thereof) labeled or conjugated with a fluorescent dye is passed through a slender flow cell along with a sheath fluid so that the cells flow in single file. The individual cells in the flow are irradiated one at a time with a light beam such as a laser beam by means of hydrodynamic focusing, and the intensity of scattered light or fluorescent light from the cells, namely light information indicative of the cells, is measured instantaneously to analyze the cells. Flow cytometry of this kind is advantageous in that a large quantity of cells can be analyzed at high speed and with great accuracy.
A known example of a cell analyzing apparatus using such flow cytometry comprises a flow cell for forming a slender stream of liquid, a light source (such as a laser) for irradiating the cells which flow through the interior of the flow cell with a light beam, a photodetector for detecting cell light information from the cells irradiated with the light beam and converting the light information into an electric signal, a signal processing circuit for amplifying, integrating and removing noise from the signal produced by the photodetector, and a computer for processing a signal, which represents the cell light information, outputted by the signal processing circuit.
For example, when subsets of lymphocytes in blood are analyzed, a monoclonal antibody labeled with a fluorescent dye is made to react with human blood, after which a hemolytic treatment is applied. The result is used as the sample.
The sample cells which flow through the flow cell are irradiated with a laser beam and four parameters, namely forward-scattered light intensity I.sub.0, 90.degree. side-scattered light intensity I.sub.90, green fluorescence intensity I.sub.g and red fluorescence intensity I.sub.r, are detected by respective detectors, as a result of which cell light information (hereinafter referred to simply as "data" where appropriate) is obtained. Since the sample contains cells such as monocytes and granulocytes in addition to lymphocytes, it is required that the data relating to the lymphocytes be discriminated and selected from the data regarding the other cells.
As shown in FIG. 11, two parameters, namely forward-scattered light intensity I.sub.0, which represents the size of cells, and 90.degree. side-scattered light intensity I.sub.90, which represents the complexity of the internal structure of cells, are used to produce a cytogram in which I.sub.90 is plotted along the horizontal axis and I.sub.0 is plotted along the vertical axis. In FIG. 11, both I.sub.90 and I.sub.0 are normalized depending upon the maximum values measured to make the maximum value on the scale 256 (eight bits). The scale is expressed in channel (ch) units. Further, b indicates the distribution of lymphocytes, c the distribution of monocytes and d the distribution of granulocytes. The distribution of debris, which is indicated at a, is composed of substances such as the membrane component of red blood cells. Often such debris is removed at the noise processing stage.
In order to analyze lymphocytes, an analytic area (a polygonal or elliptical window) B.sub.o which contains the lymphocyte distribution is set and data regarding the lymphocytes which belong to the area Bo is selected (extracted) from the measurement data. The lymphocyte data thus selected is subjected to calculation processing on the green fluorescence intensity I.sub.g and red fluorescence intensity I.sub.r, and such operations as the calculation of positive ratio and the enumeration of the lymphocyte cell count are carried out. The results are displayed on a CRT and may be printed out by a printer.
The sample often is prepared from human blood. The reason for this is that blood reflects the conditions of human disease in a variety of ways and changes often appear in the pattern of cell groups in the scattered light cytogram or fluorescent-light cytogram in flow cytometry. There are several items of information regarding the various blood cells contained in human blood. Among these items of information, the positive ratio and raw data of the counts of various cells in the overall number of cells measured are employed in the examination of lymphocyte subsets in the prior art, as mentioned above. However, depending upon the disease, the positive ratio and the raw cell count do not always reflect the conditions of the disease accurately. For example, in the case of the disease AIDS, the ratio of the number of cells contained in the CD4 cell group of antibody classification and the ratio of the number of cells contained in the CD8 cell group to the total number of white blood cells often vary greatly depending upon individual differences or from one case to another. As a consequence, the condition of a disease cannot be grasped accurately based solely upon the positive ratio or the cell count (the percentage of a specific cell count in the total number of cells).
As mentioned above, there are several items of information regarding the various blood cells contained in human blood. Since the comparison of information often is performed on the basis of the positive ratio in the examination of lymphocyte subsets, it is difficult to make a direct comparison with already existing information (the range of normal values, for example), and there is the danger that the information will not be capable of being understood from a variety of aspects. In addition, though a comparison may also be made using measured cell count as a reference, the cell counts compared are diverse and the numerical values of the compared cell counts themselves are not always sufficiently reliable. When such is the case, the objectivity, interchangeability and reliability of the information are doubtful. Furthermore, if the measured cell count does not attain a predetermined value, as is possible with a specimen from a patient in which the cells are white blood cells and the condition of the patient's disease is being monitored following administration of an anti-cancer agent, a problem of low reliability arises in terms of the reliability of analysis. In addition, even though the specimen undergoing analysis is normal, the apparent cell count will be measured on the low side if the specimen is overly diluted at the time of preparation or if it cannot be taken up sufficiently because the specimen take-up operation is performed erroneously. This will make it necessary to prepare or measure the specimen again, thereby making it difficult to improve the efficiency of automation.